POTENTIAL ADVERSE EFFECTS OF CONSUMING EDIBLE NANOEMULSION: ENHANCED PESTICIDE UPTAKE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1012328
• Grant No.: 2017-67021-26605
• Cumulative Award Amt.: $444,550.00
• Proposal No.: 2016-08782
• Project Start Date: Jun 1, 2017
• Project End Date: Aug 24, 2020
• Grant Year: 2017
• Program Code: [A1511]- Agriculture Systems and Technology: Nanotechnology for Agricultural and Food Systems

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
Food Science

Non Technical Summary
The food industry is increasingly using nanoemulsions, which consist of extremely small oil droplets dispersed in water, in food products such as dressings, sauces, dips, and beverages. Nanoemulsions are known to increase the bioavailability of beneficial hydrophobic nutraceuticals in fruits and vegetables, but they may also increase the bioavailability of harmful hydrophobic pesticides that may be on the surfaces of some produce. This project aims to understand the impact of nanoemulsion composition and structure on the bioavailability of pesticide residues on fruits and vegetables. Our central hypothesis is that certain types of nanoemulsion will increase the oral bioavailability of certain types of pesticides. This project will identify the properties of pesticides that could lead to problems due to this effect (e.g., their hydrophobicity, molecular weight, and charge characteristics). The project will be carried out in two parts:1: Influence of Nanoemulsion and Pesticide Properties on Pesticide Bioavailability. Fruits and vegetables treated with common pesticides will be mixed with nanoemulsions and then passed through a simulated gastrointestinal tract (GIT): mouth; stomach; and small intestine. The impact of nanoemulsion composition and structure on pesticide bioaccessibility, absorption, and transformation will be determined. Pesticides with different molecular weight, hydrophobicity, and charge characteristics will be studied. This research will help elucidate which types of pesticides may cause problems, and which types do not. We hypothesize that more hydrophobic pesticides (i.e, those that are more soluble in oil than in water), may be most susceptible to this effect.2: Demonstrate Impact of Nanoemulsions on Pesticide Uptake and Toxicity using Animal Feeding Studies. The ability of nanoemulsions to increase the uptake and toxicity of pesticides will be demonstrated using an animal model. Nanoemulsions and pesticides leading to the greatest increase in in vitro bioavailability in Aim 1 would be selected for the animal studies. After oral administration of pesticide-treated produce and nanoemulsions, the levels of pesticides in the systemic circulation and specific tissues of animals will be measured, as well as toxicity markers. The conditions used in these experiments would mimic those that are of commercial importance, e.g., pesticide levels on fruits and vegetables.Successful completion of this project would provide valuable information that could be used to assess any potential adverse effects of consuming nanoemulsions on human health. Moreover, it would enable foods and beverages to be formulated so that the benefits of using nanotechnology in foods and beverages could be fully realized, without causing unforeseen side-effects. This knowledge could lead to the creation of new types of food products (such as excipient nanoemulsions) that are specially designed to increase the bioavailability of healthy nutraceuticals in fresh and cooked fruits and vegetables, thereby leading to improvements in human health and wellbeing.

Animal Health Component

40%

Research Effort Categories

Basic

60%

Applied

40%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
711	1430	2000	50%
711	1122	2000	30%
711	1440	2000	20%

Knowledge Area
711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources;

Subject Of Investigation
1440 - Cole crops; 1430 - Greens and leafy vegetables; 1122 - Strawberry;

Field Of Science
2000 - Chemistry;

Keywords

pesticides

food

nanoparticles

nanoemulsions

bioavailability

toxicity

Goals / Objectives
Major GoalsOur hypothesis is that the oral bioavailability of pesticide residues on fruits and vegetables depends on the composition and structure of any nanoemulsions consumed with them, as well as the molecular and physicochemical characteristics of the pesticides themselves. Our goal is therefore to carry out systematic experiments that will enable us to identify those nanoemulsion and pesticide characteristics that may lead to an increased bioavailability of pesticides.The impact of nanoemulsion properties on the bioavailability of selected pesticides in fruits and vegetables will be established using gastrointestinal tract (GIT), cell culture, and animal models. This knowledge will then be used to elucidate the major factors impacting pesticide bioavailability. The specific aims of the project are:Aim 1: Establish Influence of Nanoemulsion and Pesticide Properties on Pesticide Bioavailability. Fresh produce (fruits and vegetables) will be collected and then treated with common pesticides at known levels. The produce will then be mixed with different types and amounts of nanoemulsion and passed through a simulated GIT consisting of mouth, stomach, & small intestine phases. The impact of nanoemulsion composition (lipid and emulsifier type) and structure (particle size distribution) on the in vitro bioavailability of the pesticides will be measured. Experiments will be carried out using pesticides with different molecular properties (molecular weight, hydrophobicity, charge). This information will enable us to determine the role of nanoemulsion and pesticide characteristics on the bioavailability of pesticide residues on produce. Emphasis will be given to establishing the underlying physicochemical mechanisms involved.Aim 2: Demonstrate Impact of Nanoemulsions on Pesticide Uptake and Toxicity using Animal Feeding Studies. The ability of nanoemulsions to increase the uptake and toxicity of pesticides will be demonstrated using an animal model. Nanoemulsions and pesticides that led to the greatest increase in in vitro bioavailability in Aim 1 would be selected for the animal studies. After oral administration of the pesticide-treated produce and nanoemulsions, the levels of pesticides in the systemic circulation and specific tissues of the test animals will be measured, as well as toxicity markers.

Project Methods
Our working hypothesis is that both the composition and structure of nanoemulsions influences the bioaccessibility, absorption, and transformation of pesticides in fruits and vegetables consumed with them. A series of nanoemulsions will therefore be prepared with a range of properties believed to impact bioavailability: oil type; oil amount; emulsifier type; and particle size distribution. These nanoemulsions will then be mixed with selected pesticide-treated fruits and vegetables. The resulting mixture will then be passed through the simulated GIT to determine the impact of specific excipient nanoemulsion properties on pesticide bioaccessibility (FB), absorption (FA), and transformation (FT). These studies will enable us establish key nanoemulsion properties contributing to pesticide bioavailability, i.e., FB, FA, or FT. Controls will be carried out by mixing a weighed amount of pure water (rather than nanoemulsion) with the fruits and vegetables and then passing the mixture through the GIT.Nanoemulsion PropertiesIn these experiments we will focus on the influence of specific nanoemulsion properties on the bioaccessibility, absorption, and transformation of pesticides from selected fruits and vegetables. Preparation conditions will be tailored to each type of fruit or vegetable studied, to mimic conditions that would be used in actual food applications. All variables will be kept constant apart from the one being studied. Initially, experiments will be carried out using a single produce type (strawberries) to establish the major factors that impact the ability of nanoemulsions to increase pesticide bioavailability.Pesticide TypeWe hypothesize that the molecular and physicochemical properties of the pesticides used will impact the ability of nanoemulsions to increase their bioavailability. In particular, we believe that hydrophobic pesticides will be much more susceptible to this effect than hydrophilic ones. For this reason, we will test a number of pesticides with different propertieson strawberries. Produce TypeWe hypothesize that the nature of the fruit or vegetable consumed will impact the efficacy of the nanoemulsions at increasing pesticide bioavailability. In these experiments, we will therefore mix four types of produce (apples, nectarines, tomatoes, and strawberries) with the same type and quantity of pesticide and pass it through the simulated GIT. In vitro GIT Model: BioaccessibilityIn this study, a simulated GIT model will be used to test different nanoemulsion formulations for their ability to increase pesticide bioavailability. This method will be based on the standardized GIT model recently proposed as a result of a consensus between numerous international scientists working in this area [59]. We recognize that this simple model cannot adequately account for the complexity of the processes occurring within the human GIT, but it does provide a useful screening tool for identifying suitable candidates that can be studied using in vivo methods. In addition, it helps provide insights into the physicochemical processes occurring within the GIT. Pesticide-treated fruits or vegetables will be converted into a puree, and then mixed with the nanoemulsions. The resultant mixtures will then be subjected to the simulated GITto elucidate the impact of nanoemulsion composition and structure of pesticide bioaccessibility. The structure and properties of the samples will be monitored as they pass through each step of the GIT model by measuring particle size, charge, and microstructureusing the methods described earlier.Pesticide Bioaccessibility: The solubilization of hydrophobic pesticides within mixed micelles in the small intestine will be taken as a measure of their bioaccessibility (FB). Consequently, we will measure the fraction of different kinds of pesticides solubilized in the micelle phase after digestion. The digested sample collected at the end of the small intestine phase will be centrifuged (16,000 g for 10 minutes), which typically leads to the formation of three phases: a lower "sediment phase" consisting of dense non-digested matter; a middle "micelle phase" consisting of mixed micelles containing solubilized hydrophobic substances; and, an upper "lipid phase" consisting of any non-digested or non-solubilized lipid phase. The micelle phase will be collected using a syringe and the concentration of solubilized pesticides will be determined appropriate methods. The fraction of the total pesticides solubilized within the micelle phase will be taken as a measure of their bioaccessibility: FB = 100 ´ mS/mT, where mS and mT are the solubilized and total masses of hydrophobic substance in the sample.?In vitro GIT Model: Absorption (FA)The fraction of a hydrophobic substance absorbed by the enterocytes lining the small intestine is a major factor determining their overall bioavailability. Consequently, we will measure the amount of solubilized pesticides that can be absorbed and transported into model intestinal epithelium cells, i.e., Caco-2 cells ). This model is widely accepted as a suitable in vitro model for studying intestinal transport processes and predicting the oral absorption of bioactive substances. We have previously used this model in studies of the bioactivity of hydrophobic food components.Initially, samples will be passed through the full GIT model, and the micelle phase will be collected (Section 3.2.2). The micelle phase will then be mixed with apical media and added to the apical compartment of the trans-well containing a monolayer of Caco-2 cells (37 ºC, 95% humidity, 5% CO2), and incubated for 4 hours, during which 100 mL of basolateral media will be collected from the basolateral compartment of the trans-well every 15 minutes. The concentration of pesticides absorbed and transported through the Caco-2 cell monolayer will then be determined using appropriate HPLC methods (depending on pesticide type). The fraction of the solubilized pesticide transported across the cells to the basolateral compartment will be taken as a measure of its absorption: FA = 100 ´ mA/mS, where mA and mS are the absorbed and solubilized masses of the pesticides, respectively.In vitro GIT Model: Transformations (FT)Changes in the chemical structure of the pesticides throughout the GIT model will be determined by isolating them from intestinal fluids using organic solvents, and then measuring their concentration and that of any metabolites in the Massachusetts Pesticide Analysis Laboratory.Demonstrate Impact of Nanoemulsions on Pesticide Uptake and Toxicity using Animal Feeding StudiesRationale and general design: The aim of this section is to use animal feeding studies to determine the impact of the nanoemulsions tested in Aim 1 on pesticide bioavailability. We have extensive experience in using animal models to study the bioavailability and bioactivity of food components [67-69]. Based on the in vitro studies, we will select nanoemulsions with compositions and structures shown to be most effective at increasing pesticide bioavailability. Then we will determine the ability of these nanoemulsions to increase the in vivo bioavailability of the selected pesticides using an animal feeding study. We will orally administer a mixture of nanoemulsion and pureed strawberries to rats, and then compare their effects on biological markers of bioavailability (i.e., levels of pesticides in blood and target tissues). The changes in the physical and chemical properties of the nanoemulsions as they pass through the animal GIT will also be measured using the methods described in Section 3.2 to determine the impact of gastrointestinal passage on their properties. The pesticide whose bioavailability is increased the most will be subject to a 13-week sub-chronic toxicity study.

Progress 06/01/17 to 05/31/21

Outputs
Target Audience:The major target audience for this research is for academic, industrial and government scientists in the agricultural, food, health care, and pharmaceutical industries.Nanoemulsions are being increasingly used in these industries to improve food quality, but they could have adverse effects such as increasing the absorption of pesticides from crops (fruits and vegetables.This grant will provide information that will determine the potential impact of these effects. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project has involved training Ph.D. students and Post Docs to prepare model food matrices, and to develop novel methods to measure the pesticide concentration on fruits and vegetables under simulated gastrointestinal conditions. How have the results been disseminated to communities of interest?The knowledge gained from the project so far has been disseminated through scientific publications, presentations at scientific meetings and research alliances, and consulting with the food industry. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We achieved the goals outlined in the project. In particular, wehave shown that nanoemulsion droplet size, composition, and emulsifier type, as well as the polarity of the pesticides, impacts the ability of nanoemulsions to increase the bioaccessiility of pesiticides on co-ingested produce. This information showed that nanoemulsions only increase the bioaccessiibility of highly hydrophobic pesticides but not hydrophilic ones. This knowledge is useful for risk assessement of the potential adverse effects of foods on the uptake of pesticdes from fresh fruits and vegetables. For most people, thepesticideresidues found on agriculture products are the main source ofpesticideexposure, which may adversely influence consumer health. The potential health hazard of residual pesticides depends on the nature of the foods they are consumed with. Studies with fat-soluble vitamins and nutraceuticals have shown that their bioaccessibility depends on food matrix composition and structure. We used anin vitromethod to investigate the influence of the dimensions of the lipid phase in model fatty foods (emulsified or bulk oil) on the bioaccessibility of various pesticides. Three pesticides that differed in their oil-water partition coefficients were selected: bendiocarb (log P= 1.7), parathion (log P= 3.8), and chlorpyrifos (log P= 5.3). These pesticides were mixed with tomato puree to representpesticide-treated agricultural products. Three model foods with different oil phase dimensions were used to represent different kinds of food product: small emulsions (d(32)= 0.14 mu m); large emulsions (d(32)= 10 mu m); and, bulk oil. Our results showed that the oil droplets underwent extensive changes as they passed through the simulated gastrointestinal tract due to changes in environmental conditions, such as pH, ionic strength, bile salts, and enzyme activities. The initial rate and final amount of lipid hydrolysis decreased with increasing lipid phase dimensions.Pesticidebioaccessibility depended on both the hydrophobicity of thepesticideand the dimensions of the co-ingested lipid droplets. The least hydrophobicpesticide(bendiocarb) had a high bioaccessibility (>95%) that did not depend on lipid phase dimensions. The more hydrophobic pesticides (parathion and chlorpyrifos) has a lower bioaccessibility that increased with decreasing lipid phase dimensions. Our results demonstrate the critical role that food structure plays on the potential uptake of pesticides from agricultural products, like fruits and vegetables. There is interest in incorporating nanoemulsions into certain foods and beverages, including dips, dressings, drinks, spreads, and sauces, due to their potentially beneficial attributes. In particular, excipient nanoemulsions can enhance the bioavailability of nutraceuticals in fruit- and vegetable-containing products consumed with them. There is, however, potential for them to also raise the bioavailability of undesirable substances found in these products, such as pesticides. In this research, we studied the impact of excipient nanoemulsions on the bioaccessibility ofpesticide-treated tomatoes. We hypothesized that the propensity for nanoemulsions to raisepesticidebioaccessibility would depend on the polarity of thepesticidemolecules. Bendiocarb, parathion, and chlorpyrifos were therefore selected because they have Log P values of 1.7, 3.8, and 5.3, respectively. Nanoemulsions with different oil contents (0%, 4%, and 8%) were fabricated to study their impact onpesticideuptake. In the absence of oil, the bioaccessibility increased with increasingpesticidepolarity (decreasing Log P): bendiocarb (92.9%) > parathion (16.4%) > chlorpyrifos (2.8%). Bendiocarb bioaccessibility did not depend on the oil content of the nanoemulsions, which was attributed to its relatively high water-solubility. Conversely, the bioaccessibility of the more hydrophobic pesticides (parathion and chlorpyrifos) increased with increasing oil content. For instance, for chlorpyrifos, the bioaccessibility was 2.8%, 47.0%, and 70.7% at 0%, 4%, and 8% oil content, respectively. Our findings have repercussions for the utilization of nanoemulsions as excipient foods in products that may have high levels of undesirable non-polar substances, such as pesticides. We were not able to do animal studies due to the cut in the originalbudget. But, good in vitro - in vivo correlations have been reported for the static simulated GIT model used, so the in vitro studies provide important information. We have published numerous research articles throughout the project and presented results at a number of conferences.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, Ruojie; Zhang, Zipei; Li, Ruyi; Tan, YB, Lv, SS, McClements, D.J. (2020). Impact of pesticide polarity and lipid phase dimensions on the bioaccessibility of pesticides in agricultural produce consumed with model fatty foods. FOOD & FUNCTION, 11, ? 6028-6037
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, Ruojie; Zhang, Zipei; Li, Ruyi; Tan, YB, Lv, SS, McClements, D.J. (2020). Impact of Pesticide Type and Emulsion Fat Content on the Bioaccessibility of Pesticides in Natural Products. MOLECULES ? 25, Article Number: 1466

Progress 06/01/17 to 08/24/20

Outputs
Target Audience:The major target audience for this research is for academic, industrial and government scientists in the agricultural, food, health care, and pharmaceutical industries.Nanoemulsions are being increasingly used in these industries to improve food quality, but they could have adverse effects such as increasing the absorption of pesticides from crops (fruits and vegetables.This grant will provide information that will determine the potential impact of these effects. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project has involved training Ph.D. students and Post Docs to prepare model food matrices, and to develop novel methods to measure the pesticide concentration on fruits and vegetables under simulated gastrointestinal conditions. How have the results been disseminated to communities of interest?The knowledge gained from the project so far has been disseminated through scientific publications, presentations at scientific meetings and research alliances, and consulting with the food industry. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We achieved the goals outlined in the project. In particular, wehave shown that nanoemulsion droplet size, composition, and emulsifier type, as well as the polarity of the pesticides, impacts the ability of nanoemulsions to increase the bioaccessiility of pesiticides on co-ingested produce. This information showed that nanoemulsions only increase the bioaccessiibility of highly hydrophobic pesticides but not hydrophilic ones. This knowledge is useful for risk assessement of the potential adverse effects of foods on the uptake of pesticdes from fresh fruits and vegetables. For most people, thepesticideresidues found on agriculture products are the main source ofpesticideexposure, which may adversely influence consumer health. The potential health hazard of residual pesticides depends on the nature of the foods they are consumed with. Studies with fat-soluble vitamins and nutraceuticals have shown that their bioaccessibility depends on food matrix composition and structure. We used anin vitromethod to investigate the influence of the dimensions of the lipid phase in model fatty foods (emulsified or bulk oil) on the bioaccessibility of various pesticides. Three pesticides that differed in their oil-water partition coefficients were selected: bendiocarb (log P= 1.7), parathion (log P= 3.8), and chlorpyrifos (log P= 5.3). These pesticides were mixed with tomato puree to representpesticide-treated agricultural products. Three model foods with different oil phase dimensions were used to represent different kinds of food product: small emulsions (d(32)= 0.14 mu m); large emulsions (d(32)= 10 mu m); and, bulk oil. Our results showed that the oil droplets underwent extensive changes as they passed through the simulated gastrointestinal tract due to changes in environmental conditions, such as pH, ionic strength, bile salts, and enzyme activities. The initial rate and final amount of lipid hydrolysis decreased with increasing lipid phase dimensions.Pesticidebioaccessibility depended on both the hydrophobicity of thepesticideand the dimensions of the co-ingested lipid droplets. The least hydrophobicpesticide(bendiocarb) had a high bioaccessibility (>95%) that did not depend on lipid phase dimensions. The more hydrophobic pesticides (parathion and chlorpyrifos) has a lower bioaccessibility that increased with decreasing lipid phase dimensions. Our results demonstrate the critical role that food structure plays on the potential uptake of pesticides from agricultural products, like fruits and vegetables. There is interest in incorporating nanoemulsions into certain foods and beverages, including dips, dressings, drinks, spreads, and sauces, due to their potentially beneficial attributes. In particular, excipient nanoemulsions can enhance the bioavailability of nutraceuticals in fruit- and vegetable-containing products consumed with them. There is, however, potential for them to also raise the bioavailability of undesirable substances found in these products, such as pesticides. In this research, we studied the impact of excipient nanoemulsions on the bioaccessibility ofpesticide-treated tomatoes. We hypothesized that the propensity for nanoemulsions to raisepesticidebioaccessibility would depend on the polarity of thepesticidemolecules. Bendiocarb, parathion, and chlorpyrifos were therefore selected because they have Log P values of 1.7, 3.8, and 5.3, respectively. Nanoemulsions with different oil contents (0%, 4%, and 8%) were fabricated to study their impact onpesticideuptake. In the absence of oil, the bioaccessibility increased with increasingpesticidepolarity (decreasing Log P): bendiocarb (92.9%) > parathion (16.4%) > chlorpyrifos (2.8%). Bendiocarb bioaccessibility did not depend on the oil content of the nanoemulsions, which was attributed to its relatively high water-solubility. Conversely, the bioaccessibility of the more hydrophobic pesticides (parathion and chlorpyrifos) increased with increasing oil content. For instance, for chlorpyrifos, the bioaccessibility was 2.8%, 47.0%, and 70.7% at 0%, 4%, and 8% oil content, respectively. Our findings have repercussions for the utilization of nanoemulsions as excipient foods in products that may have high levels of undesirable non-polar substances, such as pesticides. We were not able to do animal studies due to the cut in the originalbudget. But, good in vitro - in vivo correlations have been reported for the static simulated GIT model used, so the in vitro studies provide important information. We have published numerous research articles throughout the project and presented results at a number of conferences.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, Ruojie; Zhang, Zipei; Li, Ruyi; Tan, YB, Lv, SS, McClements, D.J. (2020). Impact of pesticide polarity and lipid phase dimensions on the bioaccessibility of pesticides in agricultural produce consumed with model fatty foods. FOOD & FUNCTION, 11, ? 6028-6037
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, Ruojie; Zhang, Zipei; Li, Ruyi; Tan, YB, Lv, SS, McClements, D.J. (2020). Impact of Pesticide Type and Emulsion Fat Content on the Bioaccessibility of Pesticides in Natural Products. MOLECULES ? 25, Article Number: 1466

Progress 06/01/19 to 05/31/20

Outputs
Target Audience:The major target audience for this research is for academic, industrial and government scientists in the agricultural, food, health care, and pharmaceutical industries.Nanoemulsions are being increasingly used in these industries to improve food quality, but they could have adverse effects such as increasing the absorption of pesticides from crops (fruits and vegetables.This grant will provide information that will determine the potential impact of these effects. Changes/Problems:The Ph.D. students working on the project (one of who was going to be a Post-Doc), were offered positions at Harvard and MIT. For this reason, we were not able to complete the proposed experiments. For this reason, we have hired a new Post Doc (Kevin Zhang) andasked for a 1-year no-cost extension to complete the experiments What opportunities for training and professional development has the project provided? The project has involved training Ph.D. students and Post Docs to prepare model food matrices, and to develop novel methods to measure the pesticide concentration on fruits and vegetables under simulated gastrointestinal conditions. How have the results been disseminated to communities of interest?The knowledge gained from the project so far has been disseminated through scientific publications, presentations at scientific meetings and research alliances, and consulting with the food industry. What do you plan to do during the next reporting period to accomplish the goals?We are carrying out experiments to determinehow pesticide type impacts the ability of nanoemulsions to increase the bioaccessibility of pesticides on model product.We are also examining the impact of essential oils (natural pesticides) on the properties of produce and their bioaccessibility using a model gastrointestinal tract.

Impacts
What was accomplished under these goals? It is important to establish the impact of food matrix effects on the bioaccessibility of co-ingested substances, such as nutraceuticals, engineered nanomaterials, pharmaceuticals, and pesticides. Recently, a standardized food model (SFM) representing a typical US diet has been developed to facilitate these investigations. This model consists of caseinate-stabilized fat droplets, free casein, pectin, starch, sucrose, and sodium chloride. The SFM was stable to creaming for 2 days, contained small particles (d approximate to 180 nm), and had a narrow particle size distribution. The SFM was mixed with pureed tomato that had been deliberately contaminated with chlorpyrifos (a hydrophobic pesticide). The resulting mixture was then exposed to an in vitro digestion model. The structural and physicochemical characteristics of the samples (microstructure, particle diameter, and surface potential) were determined after each GIT stage. Also, the bioaccessibility of both chlorpyrifos and lycopene (a hydrophobic nutraceutical) from the pesticide-contaminated tomatoes was determined after the final digestion process. For lycopene, the bioaccessibility was much lower for tomato alone (0.75%) than for tomato plus SFM (19.0%) (p < 0.05). For chlorpyrifos, the bioaccessibility was also appreciably lower for tomato alone (40.4%) than for tomato plus SFM (58.7%), but the effect was less dramatic (p < 0.05). Our results indicate that a standardized food matrix impacts the bioaccessibility of hydrophobic bioactives in fresh produce by an amount that depends on the nature of the bioactive agent. The SFM developed here may therefore be useful for screening the potential impact of food matrix effects on the bioaccessibility of ingested bioactives and other substances. In addition, we have examined the impact of pesticide type (polarity) on the bioaccessibility of pesticides on model product (tomatoes).This research has shown that the nature of the pesticide has a major impact on the ability of food matrix effects to impact bioaccessibility.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Zhang, R; & McClements DJ (2019). Impact of Pesticide Type and Lipid Content of Co-ingested Emulsions on Pesticide Bioaccessibility in Natural Products. Submitted (Dec 15, 2019) Food Research International.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang, R; Wu, W.; Zhang, Z; Lv, S, Xing, B., and McClements, D.J. (2019). Impact of Food Emulsions on the Bioaccessibility of Hydrophobic Pesticide Residues in Co-ingested Natural Products: Influence of Emulsifier and Dietary Fiber Type. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 67, 6032-6040.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang, Z; Zhang, R; McClements, DJ (2019). Establishing the impact of food matrix effects on the bioaccessibility of nutraceuticals and pesticides using a standardized food model. FOOD & FUNCTION, 10, 1375-1385.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Yao, K.; McClements, DJ; Xiang, J; et al. (2019). Improvement of carotenoid bioaccessibility from spinach by co-ingesting with excipient nanoemulsions: impact of the oil phase composition. FOOD & FUNCTION,10, 5302-5311.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Li, Ruyi; Tan, Yunbing; Dai, Taotao.., McClements, DJ. (2019). Bioaccessibility and stability of beta-carotene encapsulated in plant-based emulsions: impact of emulsifier type and tannic acid. FOOD & FUNCTION 10, 7239-7252.

Progress 06/01/18 to 05/31/19

Outputs
Target Audience:The major target audience for this research is for academic, industrial and government scientists in the agricultural, food, health care, and pharmaceutical industries.Nanoemulsions are being increasingly used in these industries to improve food quality, but they could have adverse effects such as increasing the absorption of pesticides from crops (fruits and vegetables.This grant will provide information that will determine the potential impact of these effects. Changes/Problems:Due to the reduction in funding, we will be carrying out most of the project using in vitro simulated digestion models rather than animal models What opportunities for training and professional development has the project provided? The project has involved training Ph.D. students to prepare model food matrices, and to develop novel methods to measure the pesticide concentration on fruits and vegetables under simulated gastrointestinal conditions. How have the results been disseminated to communities of interest?The knowledge gained from the project so far has been disseminated through scientific publications, presentations at scientific meetings and research alliances, and consulting with the food industry. What do you plan to do during the next reporting period to accomplish the goals?We intend to examine the impact of food ingredient properties and pesticide type on the ability of nanoemulsions to increase pesticide uptake from fresh produce

Impacts
What was accomplished under these goals? Food matrix effects impact the bioavailability and toxicity of pharmaceuticals, nutraceuticals, pesticides, and engineered nanomaterials (ENMs). However, there are currently no standardized food models to test the impact of food matrix effects using in vitro gastrointestinal models. The purpose of this study was to establish a standardized food model (SFM) for evaluating the toxicity and fate of ingested ENMs and then to assess its efficacy by examining the impact of food matrix effects on the toxicity of TiO2 nanoparticles. The formulation of the SFM was based on the average composition of the US diet: 3.4% protein (sodium caseinate); 4.6% sugar (sucrose); 5.2% digestible carbohydrates (modified corn starch); 0.7% dietary fiber (pectin); 3.4% fat (corn oil); and, 0.5% minerals (sodium chloride). The SFM consisted of an oil-in-water emulsion suitable for use in both wet and dried forms. The dried form was produced by spray drying the emulsion to improve its handling and extend its shelf-life. The particle size (D-32 = 135 nm), surface charge (-37.8 mV), viscosity, color (L*, a,* b* = 82.1,-2.5, 1.3), and microstructure of the wet SFM were characterized. The hydration properties, flowability (repose angle approximate to 27.9 degrees; slide angle approximate to 28.2 degrees), and moisture sorption isotherms of the dry SFM were comparable to commercial food powders. The potential gastrointestinal fate of the SFM was determined using a simulated gastrointestinal tract, including mouth, stomach, and small intestine steps. Conversion of the SFM into a powdered form did not impact its gastrointestinal fate. A nanotoxicology case study with TiO2 nanoparticles exposed to a tri-culture epithelial cell model showed that food matrix effects reduced ENM cytotoxicity > 5-fold. The SFM developed in the current study could facilitate studies of the impact of food matrix effects on the gastrointestinal fate and toxicity of various types of food NPs. In the typical Western diet, fruits and vegetables are often consumed with food products that exist as oil-in-water emulsions, such as creams, dressings, and sauces.Studies have shown that co-ingestion of fruits and vegetables with emulsions can increase the bioavailability of beneficial lipophilic bioactive agents, such as nutraceuticals or vitamins.Agricultural produce, however, may also be contaminated with low levels of detrimental lipophilic agents, such as hydrophobic pesticides.We therefore examined the impact of co-ingesting a common agricultural product (tomatoes) with model food emulsions on the bioaccessibility of a hydrophobic pesticide (chlorpyrifos).The impact of emulsifier type (phospholipids, whey protein, Tween 80) and dietary fiber type (xanthan, chitosan, β-glucan) on the bioaccessibility of the pesticide was measured using a simulated gastrointestinal model.Chlorpyrifos bioaccessibility depended on the type of emulsifier used to formulate the emulsions: phospholipids > Tween 80 > whey protein.Dietary fiber type also influenced pesticide bioaccessibility by an amount that depended on the nature of the emulsifier used. Overall, our results suggest that the bioaccessibility of undesirable pesticides on fruits and vegetables will depend on the nature of the emulsions they are consumed with.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: McClements, D.J. (2018). The biophysics of digestion: lipids. CURRENT OPINION IN FOOD SCIENCE 21,1-6.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: McClements, D.J. (2018). Recent developments in encapsulation and release of functional food ingredients: delivery by design. CURRENT OPINION IN FOOD SCIENCE, 23, 80-84.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Liu, X; Zhang, R; McClements, D.J. et al. (2018). Nanoemulsion-Based Delivery Systems for Nutraceuticals: Influence of Long-Chain Triglyceride (LCT) Type on In Vitro Digestion and Astaxanthin Bioaccessibility. FOOD BIOPHYSICS, 13, 412-421.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang, Z; Zhang, R; Xiao, H; Bhattacharya, K. Bitounis, D; Demokritou, P., and McClements, D.J. (2019). Development of a standardized food model for studying the impact of food matrix effects on the gastrointestinal fate and toxicity of ingested nanomaterials. NANOIMPACT, 13, 13-25.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Lv, S; Zhang, Y; Tan, H; & McClements, D.J. (2019). Vitamin E Encapsulation within Oil-in-Water Emulsions: Impact of Emulsifier Type on Physicochemical Stability and Bioaccessibility. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 67, 1521-1529
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Gao, S; Decker, EA; McClements, DJ. (2019). Molecular exchange processes in mixed oil-in-water nanoemulsions: Impact on droplet size and composition. JOURNAL OF FOOD ENGINEERING 250, 1-8.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Zhang, R; Wu, W.; Zhang, Z; Lv, S, Xing, B., and McClements, D.J. (2019). Impact of Food Emulsions on the Bioaccessibility of Hydrophobic Pesticide Residues in Co-ingested Natural Products: Influence of Emulsifier and Dietary Fiber Type. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, Under Revision

Progress 06/01/17 to 05/31/18

Outputs
Target Audience:The major target audience for this research is for academic, industrial and government scientists in the agricultural, food, health care, and pharmaceutical industries. Nanoemulsions are being increasingly used in these industries to improve food quality, but they could have adverse effects such as increasing the absorption of pesticides from crops (fruits and vegetables. This grant will provide information that will determine the potential impact of these effects. Changes/Problems:Due to the reduction in initial budget, testing of the impact of food matrix effects (lipid nanostructures) on the potential toxicity of pesticides will mainly be carried out using in vitro and cell culture methods (rather than animal studies). What opportunities for training and professional development has the project provided?The project has involved training Ph.D. students to prepare model food matrices, and to develop novel methods to measure the pesticide concentration on fruits and vegetables under simulated gastrointestinal conditions. How have the results been disseminated to communities of interest?The results of the study have been presented at scientific conferences What do you plan to do during the next reporting period to accomplish the goals?We are intend toexamine other factors that may impact the bioaccessibility of pesticides, including pesticide structure, the impact of food matrix composition (e.g., fats, dietary fibers and proteinsthat may be in actual food products). We also intend to use a combinaton of in vitro and cell culture studies.

Impacts
What was accomplished under these goals? The influence of co-ingestion of food emulsions with tomatoes on the bioaccessibility of a model pesticide (chlorpyrifos) was studied. Emulsions were fabricated with different oil contents (0 to 8%), lipid compositions (MCT and corn oil) and particle diameters (d32 = 0.17 and 10 mm). The emulsions were then mixed with chlorpyrifos-contaminated tomato puree, and the mixtures were subjected to a simulated gastrointestinal tract (GIT) consisting of mouth, stomach, and small intestine. The particle size, surface charge, and microstructure of the emulsions was measured as they passed through the GIT, and chlorpyrifos bioaccessibility was determined after digestion. The composition and structure of the emulsions had a significant impact on chlorpyrifos bioaccessibility. Bioaccessibility increased with increasing oil content, and was higher for corn oil than MCT, but did not strongly depend on oil droplet size. These results suggest that co-ingestion of emulsions with fruits or vegetables could increase pesticide bioaccessibility.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: McClements DJ, Xiao H; Demokritou P (2017). Physicochemical and colloidal aspects of food matrix effects on gastrointestinal fate of ingested inorganic nanoparticles ADVANCES IN COLLOID AND INTERFACE SCIENCE 246, 165-180.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: DeLoid GM.; Wang Y; Kapronezai K; Lorente LR, Zhang RY, Pyrgiotakis G, Konduru NV, Ericsson M, White JC; De La Torre-Roche, R; Xiao H; McClements DJ; Demokritou (2017). An integrated methodology for assessing the impact of food matrix and gastrointestinal effects on the biokinetics and cellular toxicity of ingested engineered nanomaterials. PARTICLE AND FIBRE TOXICOLOGY 14, Article 40
• Type: Journal Articles Status: Published Year Published: 2017 Citation: McClements, DJ and Xiao H (2017) Designing food structure and composition to enhance nutraceutical bioactivity to support cancer inhibition SEMINARS IN CANCER BIOLOGY 46, 215-226.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhang R; Wu, W; Zhang Z; Park YP; He, LL; Xing BS; McClements DJ (2017). Effect of the Composition and Structure of Excipient Emulsion on the Bioaccessibility of Pesticide Residue in Agricultural Products. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 65, 9128-9138.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: McClements, DJ (2018). Enhanced delivery of lipophilic bioactives using emulsions: a review of major factors affecting vitamin, nutraceutical, and lipid bioaccessibility FOOD & FUNCTION, 9, 22-41.